Question

1. Define PCR reactions, components required in each reaction and their mechanism of actions. Explain in details the steps and their thermal specifies (10 points)

Answer 1

PCR: Polymerase chain reaction

It is an in vitro technique to make multiple DNA strausing  a single DNA template. PCR has many applications these days like to make diagnosis,detect pathogens,forensic, donor selection etc.

Components

Polymerase enzyme: DNA polymerase enzyme is used in in vivo for DNA replication, likewise an enzyme called Taq polymerase is used in PCR. It is isolated from the heat resistant bacteria Thermus aquaticus. As a result taq polymerase is heat stable which make it an ideal enzyme for PCR.

Primer: It is a small oligonucleotide ,single stranded containing about 20 nucleotides in specific sequence.It provides the starting point for any DNA synthesis. Two primers are used in each reaction , which binds to each strands of the DNA template.

Template DNA: It is the DNA strand that has to be copied. It is completely of choice to select the DNA template  according to the  purpose forPCR.

Nucleotide: are the basic blocks of DNA. There are 4 types of nucleotides ATP,CTP,GTP,TTP (dNTPs) respectively. Nucleotides wil be freely available in the liquid of PCR reaction.

3 steps are present in each PCR cycle.

1.Denaturation:(960C) Increasing the temperature to separate the double stranded DNA, creating single stranded DNA templates.occur at around 960C.

2. Annealing:(55-650C) decreasing the temperature of the reaction for the primer ,each binds to the separated strands of DNA.

3: Extension(72oC) temperature is again raised so that Taq polymerase elongate by adding free deoxyribo nucleotides . Elongation happens from 5 prime to 3 prime. Taq polymerase lacks proof reading as a result the resultant Of PCR is usually visualized using gel electrophoresis.

Tgis cycle is repeated 25 to 35 times in each reaction. Not only the origins DNA strand the new strands synthesized in each reaction can participate in the next cycle.